Abstract

3227

The MCF-7/TxT50 variant was derived by continuous exposure to docetaxel alone and is positive for MDR1 (ABCB1)/P-glycoprotein. This cell line is 50x resistant to docetaxel and paclitaxel, and 8.6x and 9.8x resistant to XRP6258 and XRP9881, respectively, as determined by the SRB cell proliferation assay. These taxane derivatives also had a lower affinity for P-glycoprotein in the doxorubicin-selected MDR1 variant MES-SA/Dx5, which is 200x resistant to docetaxel and paclitaxel and 25x resistant to XRP6258 and XRP9881. Co-incubation with the P-glycoprotein inhibitor PSC-833 (PSC, 2 µM) completely restored sensitivity to wild-type levels for all taxanes. We also derived an MDR1(-) taxane-resistant variant by co-selecting with docetaxel and PSC, and these cells accumulate equivalent levels of rhodamine-123, BODIPY-paclitaxel and [3H]-docetaxel relative to wild-type cells. The MCF-7/TxTP50 variant is 9.0x resistant to docetaxel, paclitaxel, XRP6258 and XRP9881, and 5.2x resistant to epothilone B, yet retained sensitivity to the vinca alkaloids and anthracyclines. In order to assess the effects of taxanes and epothilones on tubulin polymerization, cells were exposed to drugs at various concentrations, and the proportions of polymerized and unpolymerized tubulins determined by cell lysis, electrophoresis and immunoblotting. Tubulin polymerization was significantly reduced in response to all taxanes and epothilone B treatment in MCF-7/TxTP50 relative to the wild-type MCF-7 cells, and the baseline ratio of polymerized to unpolymerized tubulin was markedly decreased in resistant versus parental cells (12% versus 36%, respectively). We have ruled out mutations in beta-1 (Class I, M40) and alpha-tubulin (Kα1) as potential resistance mechanisms. MCF-7/TxTP50 cells have significantly reduced levels of the taxane-binding target, the beta-1 tubulin isotype, and reduced levels of pan-alpha- and beta-tubulin relative to wild-type cells, and these alterations may be responsible for the 34% reduction in bound BODIPY-paclitaxel detected in this variant by FACS analysis. We are exploring the role of reduced beta-1 in taxane resistance by siRNA knockdown experiments in parental cells. Supported by a grant from Sanofi-Aventis.